Sylvia B. Smith

The localization and timing of post-translational modifications of rat rhodopsin

Rat retinas were labeled by incubation with, or intravitreal injection of, [14C]leucine along with tritiated palmitic acid, glucosamine or galactose. At selected intervals, subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted and purified by affinity chromatography and gel electrophoresis. Time courses revealed that leucine rapidly and transiently labeled the rhodopsin in the rough endoplasmic reticulum (RER), with a maximum at 1.5 hr post-injection. Subsequently, the rod outer segments (ROS) contained the labeled rhodopsin, with the ROS labeling maximally at 6-12 hr. Palmitate labeling followed the same pattern but was subject to a delay, presumably because of a large intracellular pool of the fatty acid. With palmitate the RER rhodopsin was not maximally labeled until 12 hr. The acylation of rhodopsin takes place in the RER sometime after the polypeptide has been translated but before transport to the Golgi. Glucosamine labeling was also delayed because of intracellular pools of the sugar or its metabolic derivatives. But because of secondary glycosylation in the Golgi, the rhodopsin in the ROS also labeled maximally with glucosamine at about 6 hr. Administration of [3H]galactose resulted in the labeling of rhodopsin both in vivo and in vitro, in part possibly because of its conversion to mannose and subsequent insertion into the core oligosaccharide on the RER. However, in the ROS the ratio of tritium, derived from [3H]galactose, to [14C]leucine decreased by a factor of 2 between 6 and 24 hr post-injection. Moreover, between 6 and 12 hr post-injection, labeled rhodopsin molecules in the ROS underwent a shift in mobility on gels indicative of trimming to a lower molecular weight. Thus some sugar residues may be added to the rhodopsin in the inner segment and removed in the ROS.

Transient hyperglycosylation of rhodopsin with galactose.

n Abstractn n Rhodopsins oligosaccharide chains contain predominantly two types of sugar residues: mannose and N-acetylglucosamine. In the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. Treatment of bovine rod outer segments (ROS) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. Rats injected intravitreally with [3H]galactose and [14C]leucine and maintained in darkness were killed 1 hr, 6 hr, 1, 3, or 5 days following the injection. Retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by ConA sepharose chromatography and SDS-PAGE. During the first 6 hr, galactose selectively labeled rhodopsin in the Golgi-enriched fraction resulting in increased n n n [3H]n n n [14C]n n n ratios in both Golgi and ROS. The data suggested that trimming was occurring at the transition from Golgi to ROS. Furthermore, a decrease in isotope ratio in the ROS between 6 hr and 1 day suggested further trimming of rhodopsin after membrane assembly in the ROS. Additional in vivo experiments demonstrated existing molecules of galactose on rhodopsins oligosaccharide chain using lectin affinity chromatography. Rats injected intravitreally with [35S]methionine were dark-adapted for 2 hr. Following subcellular fractionation of retinas, ConA purified rhodopsin from ROS was applied to one of two additional lectin columns: Ricinus communis agglutinin (RCA) or Griffonia simplicifolia I (GSA). Eight to nine per cent of the labeled rhodopsin was bound to and eluted from RCA, whereas none bound to GSA, indicating the presence of a β-galactoside. The RCA agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. Galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. Binding of rhodopsins oligosaccharide to RCA was abolished by pre-treatment with β-galactosidase. Decreased binding of rhodopsin to RCA was observed following intravitreal injection of castanospermine but not swainsonine. Of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. The association of galactose with rat rhodopsin appeared to be a transient one. At 2 hr, 8–9% of rhodopsin contained galactose, at 6 hr only 2·2% had galactose and by 24 hr less than 1% did. The galactose was trimmed from rhodopsins oligosaccharide presumably after its role was complete. Separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that 75% of the galactose-containing rhodopsin was in the plasma membrane and only 25% was in the discs. These findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed.n n

Addition of the chromophore to rat rhodopsin is an early post-translational event

Rat retinas were labeled either by intravitreal injection of [14C]leucine or by incubation with [3H]-leucine or [35S]-methionine. Subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted with detergent and purified by chromatography on ConA-Sepharose. A fraction enriched in rough endoplasmic reticulum (RER) and substantially free of rod outer segments (ROS) was found to contain a light-sensitive protein exhibiting the properties of rhodopsin on ConA-Sepharose or Agarose chromatography and on SDS-polyacrylamide gel electrophoresis, as well as immunologically. Intravitreal injection of [3H]-retinol also labeled the rhodopsin in the RER under conditions in which the rhodopsin in the ROS was not heavily labeled. Thus the chromophore appears to be attached to opsin shortly after the apoprotein is translated in the RER.

Synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas.

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) was quantitatively assessed in retinas of normal and rd mutant mice using short-term organ culture with [35S]methionine. Retinas were studied at ages P9-P12, time points prior to and immediately after the onset of the degeneration of the rd retina. Soluble proteins of the retinal pellet and the incubation medium were subjected to SDS-polyacrylamide gel electrophoresis. Analysis of labeled protein bands utilized a radioactivity scanning system to quantify [35S]methionine incorporation into newly synthesized IRBP. The synthesis and secretion into the incubation medium of IRBP by rd mouse retinas was comparable to normal retinas at P9-P10 but decreased by more than 50% by P12. IRBP mRNA levels were evaluated in retinas of normal and rd mice ages P7-P14. Although IRBP mRNA expression increased in the rd mouse through P10, it decreased markedly thereafter. Previously reported immunocytochemical studies suggested that IRBP was not secreted in the rd mouse retina. The results of this study indicate, however, that rd mouse retinas, when removed from the eye, have the capacity to synthesize and secrete IRBP.

Acylation and glycosylation of rhodopsin in the rd mouse

Retinas of 9-10-day-old rd and control mice were incubated for 2 hr with [14C]leucine along with either tritiated palmitic acid or galactose to investigate the acylation or glycosylation, respectively, of rhodopsin. Although other laboratories have reported that phosphorylation of rhodopsin is not detectable in rd retinas, the two post-translational modifications of rhodopsin investigated in the present work are detectable. The rod outer segments (ROS) were separated from the retinal debris containing the rough endoplasmic reticulum (RER) of photoreceptor cells by vortexing and then by linear sucrose gradients. The rhodopsin from the RER was purified by affinity chromatography and gel electrophoresis. In the acylation studies, the mean ratio of palmitate to leucine in the rd mouse was nearly twice that of controls (11.73 +/- 2.84 v. 6.81 +/- 1.04). Possible explanations for the disparity between the two groups could include: (1) a diminished internal pool size of the fatty acid; or (2) acylation of amino acids such as serine or threonine which normally are not acylated in rhodopsin. Treatment of purified rhodopsin with 1 M hydroxylamine released similar amounts of palmitate from the rd mice and controls. Hence, the higher ratio of palmitate to leucine in rd mice is apparently due to a diminished internal pool size. In the glycosylation studies, the ratio of galactose to leucine was very similar between rd mice and controls, 1.7 +/- 0.43 v. 2.47 +/- 0.74. Protein content and specific activity were determined for the crude ROS preparations and for the remaining retinal debris. Although the amount of ROS protein differed significantly between the two groups, the specific activities did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of retinal protein and phospholipid synthesis in mice exposed transplacentally to N-methyl-N-nitrosourea

Transplacental exposure to the DNA alkylating agent N-methyl-N-nitrosourea on day 16 of gestation in CD-1 albino mice induces a degeneration of the retina, the severity of which depends upon the dosage level of the drug. A 1 mg kg-1 dose provokes a progressive retinal degeneration in the offspring which begins at about 4-6 weeks of age and is characterized by gradual thinning of the retinal layers. A 15 mg kg-1 dosage of MNU provokes severe retinal dysplasia characterized morphologically by rosettes in the outer nuclear layer and loss of rod outer segments (ROS). In the present biochemical experiments, retinal protein synthesis was examined in mice 2-, 4-, and 6 weeks of age exposed to 1 mg kg-1 MNU and 2- and 5 weeks of age exposed to 15 mg kg-1 MNU. Phospholipid synthesis was examined in mice 2-, 4-, 6- and 12 weeks of age exposed to 1 mg kg-1 MNU and at 2 weeks in mice exposed to 15 mg kg-1 MNU. Retinas were incubated for 2 hr at 37 degrees C in media supplemented with either [3H]leucine for protein synthesis studies or [3H]glycerol for phospholipid synthesis experiments. Aliquots of crude ROS and the retinal debris were taken for protein determination, scintillation counting, SDS-PAGE separation of labeled opsin, phosphorus determination and TLC separation of phospholipids. Results indicated that mice exposed to 1 mg kg-1 MNU did not differ significantly from age-matched controls in these measurements, whereas mice exposed to 15 mg kg-1 MNU were significantly different from controls. These results suggest that even as early as 2 weeks of age protein and lipid metabolism are adversely affected in mice exposed to the higher dose of the alkylating agent at a critical time in retinal development, but general protein and lipid synthesis is not affected in animals exposed to 1 mg kg-1 MNU at least up to 12 weeks of age. These studies suggest further investigation of more subtle derangement in the retinal function in animals exposed to low levels of MNU.

Morphological and Biochemical Studies of the Retinal Degeneration in the Vitiligo Mouse

The vitiligo mouse has been studied since the mid 1980’s for the depigmentary condition of its skin and fur. As such, it is a promising model for the human skin disease vitiligo ( 1 ). Vitiligo may occur in isolation or in combination with other disorders, including retinal degeneration (Vogt-Koyanagi-Harada syndrome) (2). In 1988, Dr. Richard Sidman and co-workers provided a preliminary report in Mouse News Letter that the vitiligo mouse had a slow progressive retinal degeneration (3). We obtained breeding pairs from Dr. Sidman and set out to characterize the retinal degeneration in this mouse and to determine the etiology of the disease with the hope of eventually designing strategies to treat the disorder. This chapter will review our findings about the morphologic, electrophysiologic and biochemical characteristics of this mutant with particular emphasis on promising results from studies of retinoid metabolism.